Leishmaniasis is a group of diseases caused by parasite protozoa of the genus Leishmania belonging to the family of the Trypanosomatids (Killick-Kendrick, R., Med Vet Entomol 1990, 4(1): 1-24). It is transmitted by the bite of bloodsucking sand flies, grouped into the subfamily Phlebotominae, of which 30 species capable of transmitting the disease are known. There are basically three clinical presentations depending on the species involved and the host immune response, cutaneous, mucocutaneous and visceral. The latter is produced by L. infantum and is fatal without treatment. It is an endemic disease that affects 15 million people, with 2 million new cases a year in 88 countries in tropical and temperate areas (90% of them in developing countries), Desjeux, P., Clin Dermatol 1996, 14(5): 417-423.
Due to its increased prevalence in recent years it has been declared as an emerging disease by the World Health Organization and it has been described as an opportunistic parasite in immunosuppressed people, mainly in AIDS patients (Pasquau, F., et al. Eur J Clin Microbiol Infect Dis 2005, 24(6): 411-418). It is a disease that is endemic in the Mediterranean basin and in Spain visceral leishmaniasis is considered a public health problem, the dog being the reservoir of the disease; there are about 7 million animals registered in the country, and the infestation rates in this host vary between 10 and 25 percent, with higher incidence areas of up to 34% (Amela, C., et al., Eur J Epidemiol 1995, 11(2): 157-161).
Due to the limited effectiveness of treatments currently used against the disease, the toxicity of the drugs and the emergence of resistance, as well as the occurrence of recurrence, the development of an effective vaccine against leishmaniasis is necessary. In particular, the development of an effective vaccine against canine leishmaniasis will not only control the progression of the disease, but the decrease in the parasite load will interrupt the cycle of transmission between vector and humans.
From a commercial point of view, there are currently only three vaccines on the market against canine leishmaniasis: two of them sold exclusively in Brazil: Leishmune® (Fort Dodge/Pfizer), inactivated and sub-unit vaccine (purified glycoprotein fraction, mannose-fucose ligand) from Leishmania donovani (Nogueira, F. S., et al., Vaccine 2005, 23(40): 4805-10) and LeishTec® (Hertape Calier Saude Animal S.A) protein sub-units vaccine (recombinant protein A2-HIS) against visceral leishmaniasis. In Europe, the European Medicines Agency (EMEA) has recently approved the vaccine Cani Leish® (Laboratoires Bio Veto Test, Groupe Virbac), protein sub-unit inactivated vaccine (purified antigen obtained from L. infantum promastigotes) against visceral leishmaniasis, the marketing of which was initiated in 2012 (Moreno, J., et al., PLoS Negl Trop Dis 2012, 6(6): e1683). Efficacy results with these vaccines are however limited. See for instance the report prepared by the EMEA on the scientific discussion for the approval of CaniLeish® wherein it is pointed out that it has shown very limited protection of vaccinated dogs (i.e., protection is lower than 20% according to the decrease in parasite load that it induces in vaccinated animals). None of these vaccines is a DNA vaccine.
The use of the DNA vaccine pCI-neo-LACK in dogs against visceral leishmaniasis has been disclosed by Ramiro et al. (Vaccine 2003, 21(19-20): 2474-84); and Ramos, I., et al. (Vaccine 2008, 26(3): 333-44). Said vaccine was produced by cloning the sequence encoding the activated protein kinase C (LACK) antigen of Leishmania infantum. Pinto E. F. et al. (Infection and immunity 2004, 72(8), 4521-4527) disclose the intranasal vaccination of mice with pCI-neo-LACK, which reports that LACK DNA but not empty DNA promoted protective immunity. More recently, antibiotic resistance free plasmid DNA expressing LACK protein (the pORT-LACK plasmid constructed from pCI-neo-LACK by removing ampicillin and neomycin resistance genes and introducing a lac operator sequence as selectable marker) and its use against Leishmania infantum infection in an heterologous prime-boost pORT-LACK/MVA-LACK vaccination was also described (Ramos, I., et al., Vaccine 2009, 27(48): 6695-6703).
Vaccination with DNA-LACK vaccines has previously shown to be effective against L. major in the murine model (Gurunathan, S., et al., The Journal of experimental medicine, 1997, 186(7): 1137-47; Gurunathan, S. et al., Nat Med 1998. 4(12): 1409-15 and Stobie, L. et. al., Proc Natl Acad Sci 2000, 97(15): 8427-32).
There are a number of advantages of DNA vaccination relative to traditional vaccination techniques. First, it is predicted that because the proteins which are encoded by the DNA sequence are synthesised in the host, the structure or conformation of the protein will be similar to the native protein associated with the disease state. It is also likely that DNA vaccination will offer protection against different strains of a virus, by generating cytotoxic T lymphocyte response that recognise epitopes from conserved proteins. Furthermore, because the plasmids are taken up by the host cells where antigenic protein can be produced, a long-lasting immune response will be elicited. The technology also offers the possibility of combining diverse immunogens into a single preparation to facilitate simultaneous immunisation in relation to a number of disease states (see also Box 1 Saade et al., Expert Rev Vaccines 2012, 11(2):189-209).
There is a need however to improve immunogenicity of DNA vaccines, especially in humans, when compared with traditional protein-based vaccines. Many strategies have been attempted to improve DNA vaccine potency including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime-boost strategies, see Saade et al., Expert Rev Vaccines 2012, 11(2):189-209.
One approach is the use of traditional adjuvants which act as immune stimulators, as antigen delivery systems, or both. Traditional adjuvant approaches using for instance alum or lipopolysaccharide (LPS) have been mostly superseded in DNA vaccines by use of plasmid-encoded molecular adjuvants.
Despite many strategies having been attempted to improve DNA vaccine potency, there is an on-going need for research into strategies to further enhance DNA vaccines immunogenicity.
FabI gene encodes enoyl-acyl carrier protein (ACP) reductase, an enzyme which is essential for bacterial fatty acid synthesis and sensitive to triclosan which has been reported as a selection marker for molecular cloning (see for instance, Goh et al. 2008, BMC Biotechnology, 8:61). El-attar Laila M R et al. (Vaccine 2011, 30(9) 1702-1709) discloses the use of FabI from E. coli as a non-antibiotic selection marker in a DNA vaccine against a pestivirus.